Reduction of dermal scarring

ABSTRACT

Methods and compositions for reducing or inhibiting dermal scarring by expressing p21 WAF1/Cip1  in a wound site are provided.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application claims benefit of priority to U.S. ProvisionalPatent Application No. 60/524,993, filed Nov. 24, 2003, which isincorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

Excessive cutaneous scarring is an area of unmet medical need and causesfunctional, cosmetic and psychological morbidity. See. e.g., Hunt, T.K., World J Surg, 4(3): 271-7 (1980); Nicolai, J. P., et al., AestheticPlast Surg, 11(1):29-32 (1987). Clinical scar management involvesconsideration of both the continual physical assessment of the scar,including body location and the patient's previous scar history, with aclinical regimen that is often modulated over the course of treatment.Accepted conservative treatments for hypertrophic scars and keloids arelimited to surgery, corticosteroid injections, radiotherapy, siliconegel sheeting and pressure therapy. See, e.g., Mustoe, T. A., et al.,Plast Reconstr Surg, 110(2):560-71 (2002). While scar management hasrecently experienced new modalities for the physician, scar outcome isstill largely unpredictable. Treatments that specifically target thebiological mechanisms responsible for hypertrophic scars and keloidswould complement existing therapy and could improve current scaroutcome.

Cutaneous scarring is described as macroscopic disruptions of normalskin architecture and function, which arise as a consequence of woundrepair and proceeds as a fibroproliferative response. See. e.g., Clark,R. A. F., Wound Repair: Overview and General Considerations, in THEMOLECULAR AND CELLULAR BIOLOGY OF WOUND REPAIR, (Ed., R. A. F. Clark),1988, pp. 3-35. The pathogenetic and biological profile of keloids andhypertrophic scars is not fully understood. Keloids are hallmarked bygrowth beyond the margins of the original trauma site, are associatedwith familial disposition, and rarely regress. See, e.g., Tredget, E.E., Ann N Y Acad Sci, 888:165-82 (1999). Hypertrophic scars are raised,erythematous fibrous lesions which usually undergo resolution over timeand are associated with contracture of tissue. See, e.g., Tredget, E.E., Ann NY Acad Sci, 888:165-82 (1999). While keloids differ fromhypertrophic scars in genetic linkage and immunological parameters, bothare associated with fibroblast hyperproliferation and excessiveextracellular matrix (ECM) deposition. See, e.g., Rockwell, W. B., etal., Plast Reconstr Surg, 84(5):827-37 (1989); Tsao, S. S., et al.,Semin Cutan Med Surg, 21(1):46-75 (2002); Nemeth, A. J., J Dermatol SurgOncol, 19(8):738-46 (1993).

Clearly, scarring remains a problem that is difficult to avoid in manysituations. The present invention addresses this and other problems.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for reducing scarring. In someembodiments, the methods comprise administering a polynucleotidecomprising an expression cassette to skin wherein the expressioncassette comprises a promoter operably linked to a polynucleotideencoding p21^(WAF1/Cip1). In some embodiments, the polynucleotide(optionally in a vector) is administered to a wound on the skin of asubject.

In some embodiments, the DNA is administered as part of a vector. Insome embodiments, the vector is a viral vector. In some embodiments, theviral vector is an adenoviral vector. In some embodiments, theadenoviral vector is a replication deficient adenoviral vector.

In some embodiments, the administrating step results in decreasedkeloids or hypertophic scarring at the wound compared to scarring on anuntreated wound. In some embodiments, the adenoviral vector isadministered at a dose of between 10⁵ and 10⁷ particle number (PN) percm² of the wound.

In some embodiments, the vector is administered in a biocompatiblematrix. In some embodiments, the matrix comprises collagenous, metal,hydroxyapatite, bioglass, aluminate, bioceramic materials, purifiedproteins or extracellular matrix compositions. In some embodiments, thematrix is a collagen matrix.

In some embodiments, the skin is burned.

The present invention also provides pharmaceutical compositionscomprising an expression cassette and a pharmaceutically acceptableexcipient, wherein the composition is suitable for topicaladministration and the expression cassette comprises a promoter operablylinked to a polynucleotide encoding p21^(WAF1/Cip1). In someembodiments, the expression cassette (optionally a part of a vector) iswithin a biocompatible matrix.

In some embodiments, the matrix comprises a viral vector comprising theexpression cassette. In some embodiments, the viral vector is anadenoviral vector. In some embodiments, the adenoviral vector is areplication deficient adenoviral vector.

In some embodiments, the matrix comprises collagenous, metal,hydroxyapatite, bioglass, aluminate, bioceramic materials, purifiedproteins or extracellular matrix compositions. In some embodiments, thematrix is a collagen matrix.

DEFINITIONS

As used herein, “p21^(WAF1/Cip1)” refers to the wildtype full lengthp21^(WAF1/Cip1) protein, active fragments thereof, active variantsthereof, and fusions comprising the full-length p21^(WAF1/Cip1) proteinor active fragments thereof or active variants thereof, wherein thefusions retain p21^(WAF1/Cip1) activity. The wild type p21^(WAF1/Cip1)protein is a 164 amino acid protein having cell regulatory functions.See, e.g., U.S. Pat. No. 5,302,706. p21^(WAF1/Cip1) is also known in thescientific literature as p21, p21sdi, p21waf1, p21cip1 and p21pic1. Theterm “p21^(WAF1/Cip1) polynucleotide” refers to polynucleotide sequencesencoding p21^(WAF1/Cip1), including, e.g., the human wild-type proteinand homologous sequences from other organisms, as well as any mutationsor truncations thereof, or fusions that display essentially the samefunction as the wild-type p21^(WAF1/Cip1) protein.

“p21^(WAF1/Cip1)” activity refers to the ability to complement ap21^(WAF1/Cip1) mutation and to act as an inhibitor of cyclin-dependentkinase activity (Harper, J. W., et al. Cell 75:805-816 (1993)) and/orinhibit cell-cycle progression. See, e.g., Harper, J. W., et al., supra;Xiong, Y. et al. Cell 71:505-514 (1992).

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081(1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985);and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8:91-98(1994)).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

An “expression cassette” is a nucleic acid, generated recombinantly orsynthetically, with a series of specified nucleic acid elements thatpermit transcription of a particular nucleic acid in a host cell. Theexpression cassette can be part of a plasmid, virus, or other nucleicacid. Typically, the expression vector includes a promoter operablylinked to a nucleic acid to be transcribed.

A “biocompatible matrix” refers to a matrix that does not produce asignificant allergic or other adverse reaction in the host subject towhich the matrix is administered.

The term “operably linked” refers to a functional linkage between anucleic acid expression control sequence (such as a promoter, or arrayof transcription factor binding sites) and a second nucleic acidsequence, wherein the expression control sequence directs transcriptionof the nucleic acid corresponding to the second sequence.

The term “promoter” or “regulatory element” refers to a region orsequence determinants located upstream or downstream from the start oftranscription and which are involved in recognition and binding of RNApolymerase and other proteins to initiate transcription. A“constitutive” promoter is a promoter that is active under mostenvironmental and developmental conditions. An “inducible” promoter is apromoter that is active under environmental or developmental regulation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates Proliferation effects and pro-collagen I proteindetection in primary human dermal fibroblast cells afterrAd-p21^(WAF1/Cip1) treatment. The figure illustrates expression ofp21^(WAF-1/Cip-1) protein in primary human dermal fibroblast cells.Cells were treated for 48 hours with increasing concentrations of rAd,labeled with anti-p21^(WAF1/CIP1)-FITC antibody, and analyzed by FACS.In the histogram, line (A, broken line) represents staining on untreatedcells. Line (B) represents staining on cells treated with 1×10⁷ PN/mlrAd-p21^(WAF1/Cip1). Line (C) represents staining on cells treated with1×10⁸ PN/ml rAd-p21^(WAF1/Cip1). Line (D) represents staining on cellstreated with 1×10⁹ PN/ml rAd-p21^(WAF1/Cip1). Histogram isrepresentative of 3 experiments.

FIG. 1B illustrates cell cycle arrest following administration ofvarious adenoviral vectors. Cells were treated for 48 hours withincreasing concentrations of rAd (X-axis), pulse-labeled with BrdU andanalyzed by FACS. Data are plotted on the Y-axis as percentage of cellsincorporating BrdU (closed histograms). Each bar represents the mean oftriplicate wells±one standard deviation. Comparisons are significantbetween rAd-Empty at doses of 1×10⁸ and 1×10⁹ PN/ml andrAd-p21^(WAF1/Cip1) treatment group (p<0.05).

FIG. 1C illustrates PIP protein levels following administration ofvarious adenoviral vectors. Cells were incubated for 48 hours withincreasing concentrations of recombinant adenovirus (X-axis). Celllysates were harvested and analyzed for PIP by ELISA. Data are plottedas ng of PIP per ml of total lysate protein (Y-axis). Each barrepresents the mean of triplicate wells±one standard deviation.Comparisons are significant between 3×10⁹ PN/ml rAd-p21^(WAF1/Cip1)treatment group and all other groups (p<0.05).

FIG. 2 illustrates an injection schedule in the rat PVA sponge model.PVA sponges were implanted on day 0 and rAd-PDGF-B pre-treatment wasdelivered 4 days post sponge implantation. rAd-p21^(WAF1/Cip1) wasdelivered 3 days after rAd-PDGF-B pre-treatment and all sponges wereharvested 5 days later.

FIG. 3 illustrates rAd-p21^(WAF1/Cip1) treatment effects on granulationtissue fill. PVA sponges were harvested 12 days after implantation (5days after rAd-p21^(WAF1/Cip1) treatment) and Trichrome stain wasperformed. Mean percent granulation tissue fill was evaluated byquantitative image analysis as described in Materials and Methods.Comparisons are significant between rAd-PDGF-B/rAd-p21^(WAF1/Cip1) vs.rAd-PDGF-B/vehicle and rAd-PDGF-B/rAd-Empty receiving groups (p<0.001and p=0.05, respectively). However, no significant differences wereobserved between rAd-PDGF-B/rAd-p21^(WAF1/Cip1) vs. vehicle/vehicle andrAd-Empty/rAd-Empty receiving groups (p>0.3). N=7 per each treatmentgroup.

FIGS. 4A and 4B illustrate the proliferative index afterrAd-p21^(WAF1/Cip1) treatment in vivo. Mean percent of BrdU and Ki67positive cells. Immunohistochemisty was performed with anti-BrdU andKi67 antibodies in vehicle/vehicle, rAd-PDGF-B/vehicle,rAd-PDGF-B/rAd-Empty, and rAd-PDGF-B/rAd-p21^(WAF1/Cip1) treatedsponges. Comparisons are significant betweenrAd-PDGF-B/rAd-p21^(WAF1/Cip1) treatment group vs. rAd-PDGF-B/vehicleand rAd-PDGF-B/rAd-Empty groups (*p<0.01) as well asrAd-PDGF-B/rAd-p21^(WAF1/Cip1) treatment group vs. rAd-PDGF-B/vehicleand vehicle/vehicle treatment groups (**p<0.001). For BrdU, 9 fieldsfrom 3 sponges per treatment were counted. For Ki67, 4 sponges pertreatment group were counted.

FIG. 5 illustrates that p21 expression inhibits elevated scar thicknessafter the intradermal delivery of rAd-p21 in the rabbit ear excessivescarring model.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention provides methods and compositions for reducing andtreating scarring in wounded skin. The invention provides that deliveryand expression of p21^(WAF1/Cip1) to a wound site reduces thedevelopment of granulation tissue and fibroblasts. Without intending tolimit the scope of the invention, it is believed that p21^(WAF1/Cip1)inhibits the effect of inflammatory cells (e.g., neutrophils,macrophages and lymphocytes, fibroblasts, etc.) that would otherwiseresult in undue scarring and cell proliferation during wound healing.

Methods of the invention comprise delivering a polynucleotide encoding apolypeptide comprising p21^(WAF1/Cip1) or an active fragment thereof toa wound site. Expression of p21^(WAF1/Cip1) at the wound site results inwound closure with a reduction of the scarring that would otherwiseoccur. The invention is particularly useful in preventing orameliorating hypertrophic scarring and the development and growth ofkeloids.

II. Wounds

The present invention can be used to reduce scarring from any wounds tothe skin. Without limiting the scope of the invention, skin damageresulting from, e.g., burns, punctures, cuts and/or abrasions includewounds that can be treated according to the methods of the invention.The methods of the invention are useful to reduce scarring followingsurgery, including cosmetic surgery.

Ideally, wounds are treated as soon as possible after the wound hasoccurred. For example, in some cases, the wound is treated within 72,48, 24, 18, 12, 6, 3, or 1 hours after the wound occurred. In the caseof surgical scars, the methods and compositions of the present inventionare administered contemporaneously with the surgery. However, ananti-scarring effect can be realized by treatment after extended periodsfollowing the occurrence of the wound. Generally, the amount ofp21^(WAF1/Cip1) vector applied to the wounds should be increased thelonger the time period between occurrence of the wound andadministration of the vector. In the case of replication deficientadenoviral vectors where the gene is under control of a strongconstitutive promoter such as the cytomegalovirus immediate early(“CMV”) promoter as exemplified herein, the dosage typically ranges fromapproximately 1×10⁵ PN/cm² to 1×10⁹ PN/cm², 1×10⁵ PN/cm² to 1×10⁸PN/cm², or 1×10⁵ PN/cm² to 1×10⁷ PN/cm². A typical dose would beapproximately 5×10⁶ PN/cm². The above reference dose is administered toa wound site immediately (i.e., within a day) after the wound occurred.If the adenoviral vector is applied significantly later (e.g., one weekafter the wound occurred), increases in the dose of approximately10-100-fold more vector may be necessary to realize a similar effect.Typical effective p21^(WAF1/Cip1) concentrations in the tissue areapproximately 50-150 Units of activity as determined by the WAF1 ELISAkit (commercially available from Oncogene Research Products, San Diego,Calif. Cat#QIA18) with a target concentration of approximately 80-100Units of activity.

Typically, the methods of the invention inhibit or reduce scarring butdo not inhibit wound closure. p21^(WAF1/Cip1) can inhibit wound closureif expressed in sufficient amounts. For example, 2×10¹¹ PN/cm²adenoviral vectors comprising p21^(WAF1/Cip1) polynucleotides aresufficient to delay reepithelization in animal models. Additionally, atsuch doses, effects on the tensile strength of the wound are observed.Thus, it is desirable to use a sufficient amount of a vector comprisinga p21^(WAF1/Cip1) polynucleotide to inhibit or reduce scarring while notadministrating too much so as to delay re-epithlization or appreciablydecrease tensile strength.

III. Gene Delivery

To introduce a polynucleotide sequence encoding p21, it is possible toincorporate a naked plasmid comprising a promoter operably linked to ap21 polynucleotide into cells in a wound. Alternatively, thep21^(WAF1/Cip1) polynucleotide is incorporated into a viral or non-viraldelivery system and then introduced into the wound.

1. Non-Viral Delivery Systems

Non-viral delivery systems capable of directing the expression of ap21^(WAF1/Cip1) polynucleotide to the wound include expression plasmids.Expression plasmids are autonomously replicating, extrachromosomalcircular DNA molecules, distinct from the normal genome and nonessentialfor cell survival under nonselective conditions capable of effecting theexpression of a DNA sequence in the target cell. Plasmids autonomouslyreplicate in bacteria to facilitate bacterial production, but it is notnecessary that such plasmids containing the cyclin dependent kinase genereplicate in the target cell in order to achieve the therapeutic effect.The transgene may also be under control of a tissue specific promoterregion allowing expression of the transgene only in particular celltypes (e.g., inflammatory cells, including, e.g., neutrophils,macrophages and lymphocytes as well as fibroblasts and keratinocytes).Those of skill in the art will readily appreciate the variety ofexpression plasmids which may be useful in the practice of the presentinvention.

The expression plasmid may also contain promoter, enhancer or othersequences aiding expression of a p21 polynucleotide. Although one mayuse a constitutive promoter such as CMV, it may be useful to employpromoters having specific activity in the target cells such as pFascin(Sudowe, et al. Molecular Therapy 8(4):567 (2003)) and the keratin-12promoter (Ikawa, et al., Molecular Therapy 8(4):666 (2003). Induciblepromoters which are functional under certain conditions in response tochemical or other stimuli may also be employed to effectively controlexpression of p21^(WAF1/Cip1). Examples of inducible promoters are knownin the scientific literature. See, e.g., Yoshida and Hamada, Biochem.Biophys. Res. Comm. 230:426-430 (1997); Iida, et al., J. Virol.70(9):6054-6059 (1996); Hwang, et al., J. Virol 71(9):7128-7131 (1997);Lee, et al., Mol. Cell. Biol. 17(9):5097-5105 (1997); and Dreher, etal., J. Biol. Chem. 272(46):29364-29371 (1997). An example of radiationinducible promoters include the EGR-1 promoter. See, e.g., Boothman, etal. (1994) volume 138 supplement pages S68-S71

Additional genes, such as those encoding drug resistance, can beincluded to allow selection or screening for the presence of therecombinant vector. Such additional genes can include, for example,genes encoding neomycin resistance, multi-drug resistance, thymidinekinase, beta-galactosidase, dihydrofolate reductase (DHFR), andchloramphenicol acetyl transferase.

The expression plasmid containing p21^(WAF1/Cip1) polynucleotide may beencapsulated in liposomes. Liposomes include emulsions, foams, micelles,insoluble monolayers, liquid crystals, phospholipid dispersions,lamellar layers and the like. The delivery of DNA sequences to targetcells using liposome carriers is well known in the art. A variety ofmethods are available for preparing liposomes, as described in, e.g.,Szoka et al. Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos.4,394,448; 4,235,871; 4,501,728; 4,837,028; and 5,019,369. Liposomesuseful in the practice of the present invention may be formed from oneor more standard vesicle-forming lipids, which generally include neutraland negatively charged phospholipids and a sterol, such as cholesterol.

Examples of such vesicle forming lipids include DC-chol, DOGS, DOTMA,DOPE, DOSPA, DMRIE, DOPC, DOTAP, DORIE, DMRIE-HP, n-spermidinecholesterol carbamate and other cationic lipids as disclosed in U.S.Pat. No. 5,650,096. The selection of lipids is generally guided byconsideration of, e.g., liposome size, acid lability and stability ofthe liposomes in the blood stream. Additional components may be added tothe liposome formulation to increase serum half-life such aspolyethylene glycol coating (so called “PEG-ylation”) as described inU.S. Pat. Nos. 5,013,556 and 5,213,804.

In order to facilitate delivery of the therapeutic gene to a particulartissue or organ, it may be advantageous to incorporate elements into thenon-viral delivery system which facilitate cellular targeting.

2. Viral Delivery Systems

In other instances, the DNA sequence is delivered by a viral deliverysystem wherein the p21^(WAF1/Cip1) polynucleotide is incorporated into aviral genome capable of infecting the target cell and thep21^(WAF1/Cip1) polynucleotide is operably linked to expression andcontrol sequences such that the polynucleotide is expressed underappropriate conditions in the target cell. The vectors useful in thepractice of the present invention may also be derived from the viralgenomes. Vectors which may be employed include recombinantly modifiedenveloped or non-enveloped DNA and RNA viruses, preferably selected frombaculoviridiae, parvoviridiae, picornaviridiae, herpesveridiae,poxviridae or adenoviridiae. Chimeric vectors may also be employed whichexploit advantageous elements of each of the parent vector properties.See, e.g., Feng, et al. Nature Biotechnology 15:866-870 (1997). Suchviral genomes may be modified by recombinant DNA techniques to include ap21^(WAF1/Cip1) polynucleotide and may be engineered to be replicationdeficient, conditionally replicating or replication competent.Typically, the vectors are replication deficient or conditionallyreplicating. Exemplary vectors are derived from the adenoviral,adeno-associated viral and retroviral genomes. In some embodiments, thevectors are replication incompetent vectors derived from the humanadenovirus genome. The transgene may also be under control of a tissuespecific promoter region allowing expression of the transgene only inparticular cell types.

In other instances, to insure efficient delivery of the p21^(WAF1/Cip1)polynucleotide to a particular tissue or organ, it may be advantageousto incorporate elements into the viral delivery system which facilitatecellular targeting. Viral envelopes used for packaging the constructs ofthe invention can be modified by the addition of receptor ligands orantibodies specific for a receptor to permit receptor-mediatedendocytosis into specific cells (e.g., WO 93/20221, WO 93/14188; WO94/06923). In some embodiments of the invention, the DNA constructs ofthe invention are linked to viral proteins, such as adenovirusparticles, to facilitate endocytosis. See, e.g., Curiel, et al. Proc.Natl. Acad. Sci. U.S.A. 88:8850-8854 (1991). Cell type specificity orcell type targeting may also be achieved in vectors derived from viruseshaving characteristically broad infectivities by the modification of theviral envelope proteins. For example, cell targeting has been achievedwith adenovirus vectors by selective modification of the viral genomeknob and fiber coding sequences to achieve expression of modified knoband fiber domains having specific interaction with unique cell surfacereceptors. Examples of such modifications are described in Wickham, etal. J. Virol. 71 (11):8221-8229 (1997) (incorporation of RGD peptidesinto adenoviral fiber proteins); Arnberg, et al. Virology 227:239-244(1997) (modification of adenoviral fiber genes to achieve tropism to theeye and genital tract); Harris and Lemoine TIG 12(10):400-405 (1996);Stevenson, et al., J. Virol. 71(6):4782-4790 (1997); Michael, et al.Gene Therapy 2:660-668 (1995) (incorporation of gastrin releasingpeptide fragment into adenovirus fiber protein); and Ohno, et al. NatureBiotechnology 15:763-767 (1997) (incorporation of Protein A-IgG bindingdomain into Sindbis virus); and U.S. Pat. Nos. 5,721,126 and 5,559,099.Other methods of cell specific targeting have been achieved by theconjugation of antibodies or antibody fragments to the envelopeproteins. See, e.g., Michael, et al. J. Biol. Chem. 268:6866-6869(1993), Watkins, et al. Gene Therapy 4:1004-1012 (1997); Douglas, et al.Nature Biotechnology 14:1574-1578 (1996). Alternatively, particularmoieties may be conjugated to the viral surface to achieve targeting.See, e.g., Nilson, et al. Gene Therapy 3:280-286 (1996) (conjugation ofEGF to retroviral proteins).

Conditionally replicating viral vectors are used to achieve selectiveexpression in particular cell types while avoiding untoward broadspectrum infection. Examples of conditionally replicating vectors aredescribed in Bischoff, et al. Science 274:373-376 (1996); Pennisi, E.,Science 274:342-343 (1996); Russell, S. J. Eur. J. of Cancer30A(8):1165-1171 (1994).

In some instances, particularly when employing a conditionallyreplicating or replication competent vector, it may be desirable toinclude a suicide gene in the viral vector in addition to thep21WAF1/Cip1 polynucleotides. A suicide gene is a nucleic acid sequence,the expression of which renders the cell susceptible to killing byexternal factors or causes a toxic condition in the cell. A well knownexample of a suicide gene is the thymidine kinase (TK) gene (see, e.g.,U.S. Pat. No. 5,631,236 and U.S. Pat. No. 5,601,818) in which the cellsexpressing the TK gene product are susceptible to selective killing bythe administration of gancyclovir. This provides a “safety valve” to theviral vector delivery system to prevent widespread infection due to thespontaneous generation of fully replication competent viral vectors ofbroad range infectivity.

In some embodiments of the invention, the vector is derived from genusadenoviridiae. Particularly preferred vectors are derived from the humanadenovirus type 2 or type 5. Such vectors are typically replicationdeficient by modifications or deletions in the E1a and/or E1b codingregions. Other modifications to the viral genome to achieve particularexpression characteristics or facilitate repeat administration or lowerimmune response are preferred.

In some embodiments, the recombinant adenoviral vectors have complete orpartial deletions of the E4 coding region, optionally retaining (ordeleting) E4 ORF6 and ORF 6/7. The E3 coding sequence has beendemonstrated to be nonessential and may be deleted from adenoviralvectors but is preferably retained. In some embodiments, the promoteroperator region of E3 be modified to increase expression of E3 toachieve a more favorable immunological profile for the therapeuticvectors. In some embodiments, the vector used is a human adenoviral type5 vector containing a p21WAF1/Cip1 polynucleotide under control of thecytomegalovirus promoter region and the tripartite leader sequencehaving E3 under control of the CMV promoter and deletion of E4 codingregions while retaining E4 ORF6 and ORF 6/7.

In some embodiments, the adenovirus expression vector comprises apartial or total deletion of protein IX. See, e.g., U.S. patentPublication No. 2003/0091534. Other adenoviral vectors include thosedescribed in, e.g. U.S. patent Publication Nos. 2003/0192066 and2003/0157688.

IV. Pharmaceutical Formulation

The invention further provides pharmaceutical formulations comprisingthe p21WAF1/Cip1 polynucleotide in a viral or non-viral delivery systemfor administration. The compositions of the invention will be formulatedfor administration by manners known in the art acceptable foradministration to a mammalian subject, preferably a human. In particulardelivery systems may be formulated for topical administration.

The compositions of the invention can be administered in topicalformulations or polymer matrices, hydrogel matrices, polymer implants,or encapsulated formulations to allow slow or sustained release of thecompositions. Any biocompatible matrix material containing DNA encodingp21^(WAF1/Cip1) can be formulated and used in accordance with theinvention.

The gene activated matrices of the invention may be derived from anybiocompatible material. Such materials may include, but are not limitedto, biodegradable or non-biodegradable materials formulated intoscaffolds that support cell attachment and growth, powders or gels.Matrices may be derived from synthetic polymers or naturally occurringproteins such as collagen, other extracellular matrix proteins, or otherstructural macromolecules.

The type of matrix that may be used in the compositions, devices andmethods of the invention is virtually limitless and may include bothbiological and synthetic matrices. The matrix will have all the featurescommonly associated with being “biocompatible”, in that it is in a formthat does not produce an adverse, allergic or other untoward reactionwhen administered to a mammalian host. Such matrices may be formed fromnatural or synthetic materials. The matrices will typically bebiodegradeable. The matrices may take the form of sponges, implants,tubes, Telfa® pads, Band-Aid® brand adhesive bandages, bandages, pads,lyophilized components, gels, patches, artificial skins, powders ornanoparticles. In addition, matrices can be designed to allow forsustained release and/or to provide a framework into which cells maymigrate and be transduced and to provide a structural framework tofacilitate healing.

Biocompatible biodegradable polymers that may be used are well known inthe art and include, by way of example and not limitation, polyesterssuch as polyglycolides, polylactides and polylactic polyglycolic acidcopolymers (“PLGA”)(Langer and Folkman, Nature 263:797-800 (1976);polyethers such as polycaprolactone (“PCL”); polyanhydrides; polyalkylcyanoacrylates such as n-butyl cyanoacrylate and isopropylcyanoacrylate; polyacrylamides; poly(orthoesters); polyphosphazenes;polypeptides; polyurethanes; and mixtures of such polymers. Alsopolymers of polyethylene glycol (PEG), cyclodextrins and derivatizedcyclodextrins, and collagen (whether obtained from natural sources ofrecombinant) may also be employed.

One method to control the release of nucleic acids from the matrixinvolves controlling the molecular weight of the polymer as well aschemical composition of the matrix. For example, for PLGA matrices thecomposition ratio of lactic acid/glycolic acid affects the releaseperiod. Generally, a higher ratio of lactic acid/glycolic acid, such asfor example 75/25, will provide for a longer period of controlled ofsustained release of the nucleic acids, whereas a lower ratio of lacticacid/glycolic acid will provide for more rapid release of the nucleicacids.

Another particular example of a suitable material is fibrous collagen,which may be lyophilized following extraction and partial purificationfrom tissue and then sterilized. Matrices may also be prepared fromtendon or dermal collagen, as may be obtained from a variety ofcommercial sources, such as, e.g., Sigma and Collagen Corporation.Collagen matrices may also be prepared as described in U.S. Pat. Nos.4,394,370 and 4,975,527.

In addition, lattices made of collagen and glycosaminoglycan (GAG) suchas that described in U.S. Pat. No. 4,505,266 or U.S. Pat. No. 4,485,097,may be used in the practice of the invention. The collagen/GAG matrixmay effectively serve as a support or “scaffolding” structure into whichrepair cells may migrate. Collagen matrix, such as those disclosed inU.S. Pat. No. 4,485,097, may also be used as a matrix material.

Prior to the application of the matrices to the wound site, damaged skinor devitalized tissue may be removed. The matrices of the invention cancontain additional factors or compounds that improve wound healing aswell as minimizing inflammation, infection and/or hyperproliferativeresponses. Examples of such agents include silver nitrate andantibiotics.

V. Carriers

When the delivery system is formulated as a solution or suspension, thedelivery system is in an acceptable carrier, preferably an aqueouscarrier. A variety of aqueous carriers may be used, e.g., water,buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like.These compositions may be sterilized by conventional, well knownsterilization techniques, or may be sterile filtered. The resultingaqueous solutions may be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents and the like, for example, sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, etc.

The concentration of the compositions of the invention in thepharmaceutical formulations can vary widely, i.e., from less than about0.01%, usually at or at least about 2% to as much as 20% to 50% or moreby volume, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.

VII. Delivery Enhancers

The pharmaceutical formulations of the invention may optionally includeone or more delivery-enhancing agents. The term “delivery enhancingagents” includes agents which facilitate the transfer of the nucleicacid or protein molecule to the target cell. Examples of such deliveryenhancing agents include detergents, alcohols, glycols, surfactants,bile salts, heparin antagonists, cyclooxygenase inhibitors, hypertonicsalt solutions, and acetates. Alcohols include for example the aliphaticalcohols such as ethanol, N-propanol, isopropanol, butyl alcohol, acetylalcohol as described in U.S. Pat. No. 5,789,244, the entire teaching ofwhich is herein incorporated by reference. Glycols include glycerine,propyleneglycol, polyethyleneglycol and other low molecular weightglycols such as glycerol and thioglycerol.

Acetates such as acetic acid, gluconic acid, and sodium acetate arefurther examples of delivery-enhancing agents.

Examples of surfactants are sodium dodecyl sulfate (SDS) andlysolecithin, polysorbate 80, nonylphenoxypolyoxyethylene,lysophosphatidylcholine, polyethylenglycol 400, polysorbate 80,polyoxyethylene ethers, polyglycol ether surfactants and DMSO. Bilesalts such as taurocholate, sodium tauro-deoxycholate, deoxycholate,chenodesoxycholate, glycocholic acid, glycochenodeoxycholic acid andother astringents like silver nitrate may be used. Heparin-antagonistslike quaternary amines such as protamine sulfate may also be used.Cyclooxygenase inhibitors such as sodium salicylate, salicylic acid, andnon-steroidal antiinflammatory drug (NSAIDS) like indomethacin,naproxen, diclofenac may be used.

SYN3 is a surfactant-like molecule that enhances gene delivery and isdescribed in U.S. Pat. No. 6,392,069, the entire teaching of which isherein incorporated by reference for all purposes. Additional compoundsare also described in U.S. patent Publication No. 2003/0170216, hereinincorporated by reference.

The delivery of genes may also be enhanced by the use of detergents asdescribed in U.S. Pat. No. 6,165,779, the entire teaching of which isherein incorporated by reference. Detergents include anionic, cationic,zwitterionic, and nonionic detergents. Exemplary detergents include butare not limited to taurocholate, deoxycholate, taurodeoxycholate,cetylpyridium, benalkonium chloride, ZWITTERGENT™ 3-14 detergent, CHAPS(3-{(3-Cholamidopropyl)dimethylammoniol}-1-propanesulfonate hydrate,Aldrich), Big CHAP, Deoxy Big CHAP, TRITON™-X-100 detergent, C12E8,Octyl-B-D-Glucopyranoside, PLURONIC™-F68 detergent, TWEEN™ 20 detergent,and TWEEN™ 80 detergent (CALBIOCHEM™ Biochemicals).

The concentration of the delivery-enhancing agent will depend on anumber of factors known to one of ordinary skill in the art such as theparticular delivery-enhancing agent being used, the buffer, pH, targettissue or organ and mode of administration. The concentration of thedelivery-enhancing agent will be in the range of 1% to 50% (v/v),preferably 10% to 40% (v/v) and most preferably 15% to 30% (v/v).

Phosphate buffered saline (PBS) is a possible solubilizing agent forthese compounds. However, one of ordinary skill in the art willrecognize that certain additional excipients and additives may bedesirable to achieve solubility characteristics of these agents forvarious pharmaceutical formulations. For example, the addition of wellknown solubilizing agents such as detergents, fatty acid esters,surfactants may be added in appropriate concentrations so as tofacilitate the solubilization of the compounds in the various solventsto be employed. When the solvent is PBS, a preferred solubilizing agentis Tween 80 at a concentration of approximately 0.15%.

These delivery-enhancing compounds may be used alone, in combinationwith each other, or in combination with another delivery-enhancingagent.

EXAMPLES Example 1

We demonstrate that rAd-p21^(WAF1/Cip1) can effectively attenuateproliferation of human primary fibroblasts and pro-collagen type Ideposition. Additionally, we demonstrate that rAd-p21^(WAF1/Cip1)attenuates granulation tissue and ECM deposition in a rat PVA spongewound healing model. Our results suggest that exogenous expression ofp21^(WAF-1/Cip-1) is a therapeutic option to modulate excessivescarring.

METHODS AND MATERIALS

Recombinant adenovirus vector construction and purification. Therecombinant adenovirus containing human p21^(WAF-1/Cip-1) has previouslybeen described (Perkins, T. W., et al., Arch Ophthalmol. 120(7): 941-9(2002)). Briefly, the p21^(WAF-1/Cip-1) encoding region under thecontrol of the constitutive cytomegalovirus immediate early promoter(CMV) was cloned into an E1/partial E3 deleted recombinant adenovirususing methods described in Wills et. al., Hum Gene Ther. 5(9):1079-88(1994). The rAd-Empty control adenoviral vector was constructed insimilar format as rAd-p21^(WAF1/Cip1) except a transgene was notengineered into the expression cassette. rAd-PDGF-B is an E1/parital E3deleted adenovirus vector containing a CMV-PDGF-B expression cassette,cloned in the E1-deletion site. The PDGF-B cDNA was PCR amplified from ahuman placental cDNA library (Clonetech, Palo Alto, Calif.), and 100%homology was confirmed by sequence alignment to Genbank clone M12738.This cDNA was cloned into an adenovirus E1 transfer plasmid containing aCMV promoter and an E1BpIXpoly-A expression cassette. Homologousrecombination in E. coli strain BJ5183 by the method of Chartier et al.was used to generate infectious viral DNA which was subsequentlytransfected into human kidney 293 cells to generate and propagate virus(Chartier, C., et al., J Virol, 70(7):4805-10 (1996)). Virus particleswere purified by column chromatography (Shabram, P. W., et al., Hum GeneTher, 8(4):453-65 (1997)), quantified, and dosed by particle number (PN)based upon guidance from the Food and Drug Administration (Guidance forHuman Somatic Cell Therapy and Gene Therapy, Center for BiologicsEvaluation and Research, March 1998).

Cells. Normal adult human dermal fibroblast cells were obtained fromCambrex Bio Science (Rutherford, N.J.) and maintained in recommendedgrowth media. Experiments were performed with cells at passage number≦4.

Adenovirus infections and bromodeoxyuridine pulse-labeling of cells.Cells were synchronized in G0/G1 by plating in Fetal Bovine Serum (FBS)deficient media for two days and were subsequently treated with varyingdoses (1×10⁸-3×10⁹ PN/ml) of either rAd-p21^(WAF)1/Cip1 or rAd-Empty inFBS deficient media. After 24 hrs, media was removed and mediacontaining 20% FBS was added to release cells from G0/G1 arrest. Cellswere pulse-labeled at 24 hours post-release with 10 μM bromodeoxyuridine(BrdU; Boeheringer-Mannheim, Indianapolis, Ind.) for 4 hours andharvested for bivariate BrdU/DNA flow cytometric analysis by fixation in70% ethanol, followed by digestion with 0.08% pepsin for 30 min at 37°C. Cells were centrifuged at 1500 RPM, resuspended in 2 N HCl andincubated at 37° C. for 20 minutes. 1 M sodium borate was added andcells were washed in IFA/Tween 20 (0.01 M HEPES, 0.005% sodium azide,0.5% Tween 20, 5% FBS, 0.15 M NaCl), and incubated for 30 minutes with a1:10 dilution of anti-BrdU antibody (Becton-Dickinson, Franklin Lakes,N.J.) without Tween 20. Finally, cells were washed in IFA/Tween 20,incubated in IFA/Tween 20/RNase for 15 minutes at 37° C., stained withpropidium iodide (50 μg/ml) and analyzed on the FL-1 channel by a FACScan flow cytometer (Becton Dickinson) using the CellQuest (BectonDickinson) software.

Enzyme linked immunoassay for detection of human procollagen type 1C-peptide (PIP). Cells were plated in complete media containing 10% FBS,grown to confluency and infected with adenovirus constructs in mediadeficient of FBS for 24 hours. Cells were then washed and cultured for24 hours in media without FBS prior to PIP analysis. Detection of PIPwas evaluated by ELISA (TaKaRa Bio Inc., Japan) on cell lysates with1×10⁶ cells, according to the manufacturers instructions.

p21^(WAF-1/CIP-1) detection by FACS. Cells were fixed in 75% ethanol/PBSfor 30 minutes at 4° C. and blocked for non-specific antibody bindingwith 0.1% BSA/PBS at 37° C. for 30 minutes. 2 μg/mlanti-p21^(WAF-1/CIP-1) antibody conjugated with FITC (Ab-1, Oncogene,San Diego, Calif.) was incubated with cells for 60 minutes at roomtemperature. Cells were washed in 0.1% BSA, resuspended in PBS andanalyzed by FACS on the FL-1 channel.

PVA sponge model. Animal care and experiments were conducted inaccordance with the National Institutes of Health Guide for the Care andUse of Laboratory Animals and were approved by appropriate reviewcommittees. Male Sprague-Dawley rats (Harlan, Indianapolis, Ind.)weighing 350-400 grams, were anesthetized with ketamine/xylazine and sixfull thickness, 5 mm linear incisions were made on the ventral surfaceof each rat. A single sterile polyvinyl alcohol (PVA) sponge (Grade 3:12.7 mm×3 mm; M-PACT, Eudora, Kans.) was inserted subcutaneously intoeach incision and closed with wound clips. Four days after spongeimplantation, 1×10⁹ PN of rAd-PDGF-B or rAd-Empty formulated in 200 μlof collagen solution (Cohesion Technologies, Palo Alto, Calif.) or 200μl vehicle control were injected into the interior of each sponge. Threedays after the first injections, 1×10⁹, 1×10¹⁰ or 5×10¹⁰ PN ofrAd-p21^(WAF1/Cip1) formulated in 100 μl of vPBS (PBS, 3% sucrose v/v)was injected into each sponge. Animals were euthanized 5 days after thesecond injection and central portions of each sponge were harvested,fixed in 4% paraformaldehyde, paraffin embedded, and sectioned 6-μmthick. For proliferative index studies, 50 mg/kg of BrdU(Calbiochem-Novabiochem Corp., San Diego, Calif.) was injected into ratsintraperitoneally 24 hours prior to euthanasia.

Immunohistochemistry. For p21^(WAF-1/Cip-1) protein detection, 6-μm, PVAsponge paraffin sections were immersed in −20° C. ethanol/acetic acid(2:1) for 10 minutes, followed by antigen retrieval in high pH buffer(Dako, Carpinteria, Calif.) with steam for 20 minutes. Endogenousperoxidase was quenched by incubation with 3% (v/v) H₂O₂. Slides wereblocked in 20% (v/v) goat serum and then incubated with mouse monoclonalanti-human p21^(WAF-1/Cip-1) (1:100, BD-PharMingen, San Diego, Calif.)antibody for one hour. After washing in PBS (3×5 minutes), slides wereincubated with biotinylated goat anti-mouse IgG secondary antibody(1:200, Zymed, San Francisco, Calif.) for 30 minutes. Slides were rinsedin PBS (3×5 minutes), incubated with Steptavidin/HRP conjugate (Dako)for 10 minutes and developed with AEC chromagen/substrate (Dako)followed by a hematoxylin counterstain. For BrdU detection, a combinedprotocol and reagents from Zymed's BrdU staining kit and VectastainElite ABC kit (Vector Labs, Burlingame, Calif.) was used. Briefly,tissue sections were placed on sialyted glass slides and microwaved at50% power for 5 minutes. Sections were steamed for 20 minutes inpre-heated citrate buffer (pH 6.0), rinsed, and endogenous peroxidasewas quenched with 3% (v/v) H₂O₂ for 10 minutes. The Zymed kit protocolwas followed up to and including incubation with biotinylated mouseanti-BrdU primary antibody. Slides were subsequently rinsed in PBS,incubated with Vectastain Elite ABC reagent for 30 minutes, rinsed againand developed with Vector Nova red chromagen/substrate (Vector Labs) for5-15 minutes. For Ki67 detection, paraffin tissue sections weremicrowaved at maximum power in 10 mM citrate buffer (pH 6.0), washed inPBS (3×5 minutes) and blocked with 20% (v/v) goat serum for 30 minutes.Sections were incubated with mouse anti-human Ki67 (1:100; PharMingen)primary antibody for 1 hour, rinsed in PBS (3×5 minutes) and reactedwith a 1:200 dilution of biotinylated goat anti-mouse IgG (PharMingen)for 30 minutes. Diaminobenzidine (DAB; Vector Labs) was used forchromagen detection of Ki67 positive cells per kit instructions followedby counterstain with hematoxylin.

Quantitative image analysis. To evaluate the extent of granulationtissue fill, histological sections of PVA sponges were processed usingMasson Trichrome staining method. Computer-assisted quantitativeanalysis was performed using Image Pro Plus™ software (MediaCybernetics, Silver Spring, Md.) with calibrated digital photographsacquired with a Nikon E600 microscope and a 4× Plan-Fluor objective(Nikon USA, Melville, N.Y.). Analysis consisted of quantifyinggranulation tissue area within the interior of the sponge divided by theentire sponge area interior multiplied by 100 for percent granulationtissue fill. Sample size consisted on average of 6 sponges per treatmentgroup. For proliferative index in vivo, images from BrdU and Ki67immunohistochemical sections were digitally acquired with a 40× or 4×microscope objective and imported into Adobe Photoshop 5.0 software(Adobe Systems Inc., San Jose, Calif.). Positive cell counts (red orbrown) and total cell counts (purple nuclei) were scored for each fieldobserved. For BrdU, three-400× fields (minimum of 1500 cells) per tissuesection were counted from each group. For Ki67, 3 entire tissue sectionsper group (minimum of 13,000 cells/section) were evaluated. Nuclearstaining of BrdU or Ki67 regardless of staining intensity, wasconsidered positive. Percent proliferative index was calculated bydividing the red or brown cell population count by the red or brown pluspurple cell population multiplied by 100.

Statistical analyses. Data are presented as arithmetic means±SD or ±SEM,where noted. Statistical analysis of data was conducted using unpairedstudent t-test (StatView, SAS Institute Inc, Cary, N.C.). Differenceswere considered significant at p≦0.05

RESULTS

Human Dermal Fibroblast Cells Express Exogenous p21^(WAF-1/Cip-1)Protein and Exhibit Cell Cycle Arrest in Response to rAd-p21^(WAF1/Cip1)

p21^(WAF-1/Cip-1) gene expression was assessed in human primary dermalfibroblasts cells treated with increasing doses of rAd-p21^(WAF1/Cip),or rAd-Empty for 48 hours. In response to rAd-p21^(WAF1/Cip1) but notrAd-Empty, the expression of p21^(WAF-1/Cip-1) increased in a dosedependent manner, as determined by FACS analysis (FIG. 1A). In responseto 1×10⁸ PN/ml, p21^(WAF1/Cip1) expression increased over untreatedcells from 4.1±1.0 (untreated) to 6.0±0.2 (p=0.005). In response to thehighest dose of 1×10⁹ PN/ml rAd-p21^(WAF1/Cip1), expression increasedover untreated cells from 4.1±1.0 to 35.1±1.5 (p=0.001). Therefore,p21^(WAF-1/Cip-1) could be efficiently expressed in primary dermalfibroblasts in a dose dependent manner.

Next, we performed in vitro dose response studies to determine ifexogenous p21^(WAF-1/Cip-1) expression could induce cell cycle arrest inhuman dermal fibroblasts. Cells treated with 1×10⁷ to 1×10⁹ PN/mlrAd-p21^(WAF1/Cip1) showed a dose dependent reduction in cellproliferation as measured by BrdU incorporation and FACS analysis (FIG.1B). The percent of cells in S-phase decreased from an average of64.4±5.7% in the untreated cell population, to 20.6±2.6% in response to1×10⁸ PN/ml of rAd-p21^(WAF1/Cip1). At the highest dose ofrAd-p21^(WAF1/Cip1), 1×10⁹ PN/ml, a decrease in percent of cells inS-phase was observed with only 0.6±0.1% positive cells detected.Detectable cell cycle inhibition was observed with rAd-Empty at the twohighest doses. Specifically, at 1×10⁸ PN/ml of rAd-Empty, 56.9±1.0% ofcells incorporated BrdU, while at 1×10⁹ PN/ml, 34.4±10.7% of cellsincorporated BrdU. Attenuation of proliferation in response to highdoses of control adenoviruses has been previously described (Brand, K.,et al., Gene Ther, 6(6):1054-63 (1999)). BrdU incorporation wassignificantly reduced when rAd-p21^(WAF1/Cip1) was compared to rAd-Emptyat 1×10⁸ and 1×10⁹ PN/ml (p<0.05). These data suggest that while highdoses of recombinant adenovirus treatment induce generalanti-proliferative effects, we have demonstrated ap21^(WAF1/Cip1)-specific dose dependent reduction in proliferativeresponse with rAd-p21^(WAF1/Cip1) in wound target cells.

Human Dermal Fibroblast Cells Decrease Production of PIP in Response torAd-p21^(WAF1/Cip1).

To determine if the PIP peptide was reduced after delivery ofp21^(WAF1/Cip1), human dermal fibroblasts were treated with increasingdoses of rAd-p21^(WAF1/Cip1), or rAd-Empty for 48 hours (FIG. 1C).ELISAs on lysates from equal number of cells were performed to quantifyintracellular PIP levels. The data showed greater then 2-fold reductionin PIP after human dermal fibroblasts were treated withrAd-p21^(WAF1/Cip1) as compared to control groups. A decrease in PIP wasdetected at the highest concentration of rAd-p21^(WAF1/Cip1) treatment(3.0×109 PN/ml) when compared to untreated cells (60.0±6.3 ng/mlvs.165.1±9.0 ng/ml of protein, respectively. The highest dose ofrAd-Empty treatment showed 170.2±10.3 ng/ml of intracellular PIP,unchanged from uninfected cells. Our previous studies have shown that100% of human dermal fibroblasts are positive for transgene expressionin this assay system as evaluated by FACS analysis (data not shown).Apoptosis assays by Annexin V staining and FACS were performed todetermine the viability of cells and the data demonstrated that cellswere not apoptotic (data not shown). These data show that anextracellular-associated peptide is attenuated after rAd-p21^(WAF1/Cip1)treatment.

rAd-p21WAF1/Cip1 Attenuates Granulation Tissue Following rAd-PDGF-BStimulation in vivo.

To determine the effect of rAd-p21^(WAF1/Cip1) on granulation tissue invivo, a rat PVA sponge model was used (Buckley, A., et al., Proc NatlAcad Sci USA, 82(21):7340-4 (1985)). PVA sponges in rats were injectedwith 1×10⁹ PN/sponge of rAd-PDGF-B as a stimulator of granulation tissue(Liechty, K. W., et al., J Invest Dermatol, 113(3):375-83 (1999)) andthree days later rAd-p21^(WAF1/Cip1) was administered at 5.0×10¹⁰PN/sponge (FIG. 2). rAd-Empty virus was also delivered to sponges at thesame dose levels as rAd-PDGF-B and rAd-p21^(WAF1/Cip1) to control forgeneral effects that recombinant adenovirus may contribute to this modelsystem. There was reduced granulation tissue both in quantity and celldensity in rAd-PDGF-B/rAd-p21^(WAF1/Cip1) treated sponges as assessed byTrichrome stain within PVA sponges at day 5 post the rAd-p21^(WAF1/Cip1)delivery (FIG. 3). The vehicle/vehicle (FIG. 3) and rAd-Empty/rAd-Emptygroups (not shown) had similar granulation tissue morphology asrAd-PDGF-B/rAd-p21^(WAF1/Cip1) treatment groups (FIG. 3). Consistentwith published reports, the highest granulation tissue fill was observedin rAd-PDGF-B/vehicle treatment group (77%; FIG. 3), demonstrating thatthe stimulator, PDGF-B enhanced growth response in this model (Liechty,K. W., et al., J Invest Dermatol, 113(3):375-83 (1999)). We observed 53%granulation tissue fill in rAd-PDGF-B/rAd-Empty, 28% inrAd-PDGF-B/rAd-p21^(WAF1/Cip1), 24% in vehicle/vehicle, and 18% inrAd-Empty/rAd-Empty treatment groups (FIG. 3). Compared torAd-PDGF-B/vehicle and rAd-PDGF-B/rAd-Empty treatment, rAd-PDGF-Bfollowed by rAd-p21^(WAF1/Cip1) treatment induced a 2.7- and 1.9-foldattenuation in granulation tissue fill, respectively (p<0.001 andp=0.05). In contrast, no significant differences were observed betweenvehicle/vehicle, rAd-Empty/rAd-Empty, and rAd-PDGF-B/rAd-p21^(WAF1/Cip1)treatment groups (p>0.3). Vehicle/vehicle, rAd-PDGF-B/vehicle, andrAd-PDGF-B/rAd-empty treatment groups were all significantly differentfrom each other (p<0.001 and p=0.01, respectively). In a separate study,a dose response with rAd-p21^(WAF1/Cip1) was performed between 1×10⁹ and5×10¹⁰ PN/ml (Table 1). There was a 23% drop in granulation content whencompared to maximum fill in rAd-PDGF-B/vehicle treatment at 1×10⁹, 37%decrease at 1.0×10¹⁰ and 47% reduction at 5×10¹⁰ PN/ml. These datademonstrate a quantitative and qualitative reduction in granulationtissue after rAd-p21^(WAF1/Cip1) treatment. TABLE 1 Dose responsepercent decrease of granulation tissue fill within PVA sponges afterrAd-p21^(WAF1/Cip1) treatment. Treatment Groups 1st injection rAd-PDGF-BrAd-PDGF-B rAd-PDGF-B rAd-PDGF-B rAd-p21^(WAF1/Cip1) rAd-p21^(WAF1/Cip1)rAd-p21^(WAF1/Cip1) 2nd injection vPBS (1 × 10⁹) (1 × 10¹⁰) (5 × 10¹⁰)Granulation area^(a) 100% 77% 63% 54% (percent fill)Trichrome stained PVA sponge sections were analyzed by computer assistedimage analysis for granulation fill area within sponges. Percent fillwas calculated as area of granulation tissue/total area analyzed × 100.Refer to FIG. 2 for injection schedule timing. All rAd-PDGF treatmentswere dosed at 1 × 109 PN/sponge. Each treatment group represents themean of 6 individual sponges and a minimum of 22 measurements per group.Data is representative of two separate experiments.^(a)Normalized to % of maximum fill treatment (77%, rAd-PDGF-B/vPBStreatment).Expression of p21^(WAF-1/Cip-1) Protein in vivo.

To validate transduction in vivo and link p21^(WAF-1/Cip-1) expressionwith reduction of granulation tissue, we used an anti humanp21^(WAF-1/Cip-1) specific antibody to identify p21^(WAF-1/Cip-1)expressing cells in the rat PVA sponge model. Five days after treatmentwith rAd-p21^(WAF1/Cip1), p21^(WAF-1/Cip-1) protein was localized withingranulation tissue cells, which morphologically resembled inflammatorycells (FIG. 5C, small arrowhead) and fibroblasts-like cells. We did notobserve p21^(WAF-1/Cip-1) positive stained cells in either thevehicle/vehicle or rAd-PDGF-B/vehicle treatment groups. While thepredominant population of p21^(WAF-1/Cip-1) expressing cells exhibitedintense staining 5 days after rAd-p21^(WAF1/Cip1) administration, wehave currently not defined the peak response and duration ofp21^(WAF-1/Cip-1) protein expression in this model. However,rAd-p21^(WAF1/Cip1) expression by RT-PCR peaked within one week andpersisted beyond 30 days in a rabbit model of glaucoma filtrationsurgery (Perkins, T. W., et al., Arch Ophthalmol, 120(7):941-9 (2002)).These data support a link between reduction in granulation tissue andp21^(WAF-1/Cip-1) expression in vivo.

Proliferation Index is Attenuated After rAd-p21WAF1/Cip1 Treatment invivo.

To determine the proliferation status of granulation tissue afterrAd-p21^(WAF1/Cip1) delivery, BrdU and Ki67 immunohistochemical stainingwas performed on PVA sponge tissue. The percent of BrdU and Ki67positive cells in vehicle/vehicle, rAd-PDGF-B/vehicle,rAd-PDGF-B/rAd-Empty, and rAd-PDGF-B/rAd-p21^(WAF1/Cip1) treatmentgroups are presented in FIG. 4. The highest BrdU staining was observedin rAd-PDGF-B/vehicle and rAd-PDGF-B/rAd-Empty treatment groups (25% and24%, respectively), demonstrating that rAd-PDGF-B promoted tissueproliferation and also suggesting that rAd-Empty treatment had minimalimpact on proliferative status in vivo. The lowest percent BrdU stainedcells was identified in rAd-PDGF-B/rAd-p21^(WAF1/Cip1) treatment group(9%). BrdU incorporation in the rAd-PDGF-B/rAd-p21^(WAF1/Cip1) treatmentgroup was significantly lower when compared to rAd-PDGF-B/vehicle andrAd-PDGF-B/rAd-Empty (p<0.01 for both comparisons). In addition, thevehicle/vehicle treatment group had 2-fold greater number of BrdUpositive cells when compared to rAd-PDGF-B/rAd-p21^(WAF1/Cip1) treatmentgroup (18% vs. 9%, respectively).

While BrdU incorporation requires S phase initiation, Ki67 or Mib1antigen is expressed in all cell cycle phases except G₀ (Barnard, N. J.,et al., J Pathol, 152(4):287-95 (1987). The analysis of Ki67 stainingshowed similar percentage of positve cells as BrdU staining (FIG. 4B).Proliferation indices of vehicle/vehicle and rAd-PDGF-B/vehicletreatment groups were 22% and 34%, respectively. In contrast,rAd-PDGF-B/rAd-p21^(WAF1/Cip1) treatment groups revealed significantlyless percent proliferating cells (11%) when compared torAd-PDGF-B/vehicle and vehicle/vehicle treatment groups (p<0.001 for allcomparisons). These data demonstrate that rAd-p21^(WAF1/Cip1) treatmentattenuates granulation tissue in vivo by reducing cell proliferation.

SUMMARY

The etiologies of hypertrophic scars and keloids are unknown but likelyarise from dysregulation in the normal wound healing response. Normalwound healing proceeds as a fibroproliferative response that developsinto a fibrotic scar. Importantly, even in the best circumstances, theinjury site is “patched” rather than “restored”, and both form andfunction are affected by the mechanisms responsible for replacementverses tissue regeneration. The normal wound healing cascade iscomprised of 3 temporal, overlapping responses including inflammation,proliferation and remodeling phases. In all phases, there exists anequilibrium between catabolic and metabolic processes involvinggrowth-promoting factors and factors responsible for down-regulating theproliferative response. While significant progress has been made toelucidate the factors involved in stimulating a wound to heal, far lesshas been made with regard to the molecular processes, including cellcycle regulation and programmed cell death involved in normal woundhealing responses. The data presented herein, underscores the importantrole that cell cycle regulation has on the processes involved in woundrepair and scar formation.

Our studies demonstrate that the cell cycle of human primary dermalfibroblasts can be efficiently inhibited in human primary dermalfibroblasts with p21^(WAF-1/Cip-1) delivered via a recombinantadenovirus. We showed a dose dependent increase in p21^(WAF-1/Cip-1)protein expression that correlates with a dose dependent decrease inproliferative status as evidenced by BrdU staining in vitro. There weredetectable anti-proliferative effects observed with control adenovirustreatment at the highest dose, but propidium iodide staining revealedthat these cells had accumulated in G2/M, rather than G1, as in the caseof the cells treated with rAd-p21^(WAF-1/CIP-1) (data not shown). Theobservation that transduction with a high dose of recombinant adenovirusvector alone causes attenuation of cellular proliferation has beenpreviously described and several studies report anti-tumor effects withhigh doses of recombinant adenovirus containing reporter genes (Erhardt,J. A. and R. N. Pittman, Oncogene, 16(4):443-51 (1998); Pierce, G. F.,et al., J Exp Med. 167(3):974-87 (1988) Teramoto, S., et al., Hum GeneTher. 6(8):1045-53 (1995)). Increasing levels of cellularp21^(WAF-1/Cip-1) protein correlate to a decrease in proliferativeresponse in human dermal primary fibroblasts.

The excessive accumulation and disorganization of extracellular matrix,namely collagen, is a hallmark of keloids and hypertrophic scars(Rockwell, W. B., et al. Plast Reconstr Surg, 84(5):827-37 (1989)). Thetwo chemotherapeutic agents, mitomycin C and doxorubicin, have beenreported to inhibit the wound healing response with mechanisms of actionincluding reduction of ECM and cytotoxicity (Saika, S., et al.,Ophthalmic Res. 29(2):91-102 (1997)). Our studies show that elevatedlevels of p21^(WAF-1/Cip-1) in dermal primary fibroblasts reduced PIPlevels in vitro. Interestingly, PIP levels were attenuated but notablated suggesting that basal levels of PIP production are beingmaintained in viable cell populations. In contrast, while both mitomycinC and doxorubicin decreased PIP secretion, a dose dependent reduction incell viability was observed and is likely causative of wound dehiscenceobserved in a wound healing animal model (Saika, S., et al., OphthalmicRes. 29(2):91-102 (1997)). Further, PIP levels were not affected byrAd-Empty treatment suggesting that PIP attenuation wasp21^(WAF-1/Cip-1) specific. Transduced cells were transcriptionallyactive (data not shown) demonstrating that reduction in PIP is not aresult of general transcriptional depression within the cell. Wehypothesized that reduction of extracellular matrix production as aresult of exogenously expressed p21^(WAF-1/Cip-1) would have attenuatingeffects on granulation tissue production in vivo.

An animal model which exactly simulates the biochemical andpathophysiolgical parameters of human keloids and hypertrophic scarsdoes not exist. In this report, we used an animal model system toaddress the effects of elevated p21^(WAF-1/Cip-1) on granulation tissuein vivo. Granulation tissue is composed of fibroblasts, new capillaries,inflammatory cells and extracellular matrix and is a necessary andrequired element of wound repair. Disruption of the normal temporal andspatial formation of granulation tissue is implicated as a causativeeffect in hypertrophic scars and keloids. PDGF-BB is a potent stimulatorof granulation tissue formation and recent reports have demonstratedpotent pro-wound healing effects in wound impaired models (Liechty, K.W., et al., J Invest Dermatol. 113(3):375-83 (1999); Pierce, G. F., etal., J Exp Med. 167(3):974-87 (1988); Pierce, G. F., et al., J CellBiochem. 45(4):319-26 (1991); Doukas, J., et al., Hum Gene Ther.12(7):783-98 (2001)). Interestingly, addition of PDGF-BB to a scarlessfetal model results in wound fibrosis and elevated levels of PDGF-BBhave been associated with liver cirrhosis (Haynes, J. H., et al., JPediatr Surg. 29(11):1405-8 (1994); Peterson, T. C. and R. A. Isbrucker,Hepatology 15(2):191-7 (1992)). We used rAd-PDGF-B to enhance cellularinflux, proliferation and granulation tissue deposition and thenfollowed with rAd-p21^(WAF1/Cip1) treatment in the rat PVA sponge modelto determine if p21^(WAF-1/Cip-1) could attenuate these stimulatoryeffects in vivo.

Our results show that rAd-p21^(WAF1/Cip1) attenuated granulation fillboth qualitatively and quantitatively when compared to rAd-PDGF-Btreatment alone. The diminution of granulation fill after rAd-Emptytreatment is consistent with our in vitro results and pales in effectwhen compared to rAd-p21^(WAF1/Cip1) treatment. Initially, wehypothesized that rAd-Empty treatment alone may interfere withgranulation tissue outcome via the well-documented immunomodulatoryeffects of the rAd delivery vehicle on the host (Nielsen, L. L., OncolRep 7(1):151-5 (2000); Kajiwara, K., et al., Hum Gene Ther,. 8(3):253-65(1997); St George, J. A., et al., Gene Ther, 3(2):103-16 (1996); Brody,S. L., et al., Hum Gene Ther, 5(7): p. 821-36 (1994)). We repeatedlyobserved that the stimulatory and inhibitory effects of PDGF-BB andp21WAF-1/Cip-1 respectively, modulate granulation tissue activity overand above recombinant adenovirus derived responses. Critical to thisobservation is our demonstration of a rAd-p21^(WAF1/Cip1) dose dependentattenuation of granulation tissue in vivo, further supporting genespecific activity in this model system.

We were able to demonstrate human p21^(WAF-1/Cip-1) protein expressionin sponges treated with rAd-p21^(WAF1/Cip1), thus linking humanp21^(WAF-1/Cip-1) with reduction of granulation tissue. We also showedreduced proliferation by two separate assays in p21^(WAF-1/Cip-1)treated sponges, supporting the direct anti-proliferative effects ofp21^(WAF-1/Cip-1) in vivo. Cell-specific protein expression was notdetermined in these studies but morphologically, our results suggestthat both macrophages and fibroblasts can express exogenousp21^(WAF-1/Cip-1) protein.

Skin, the largest organ of the body, offers local-regional delivery withlimited systemic exposure, is accessible and can be non-invasivelyexamined. Both viral and non-viral approaches have demonstrated genetransfer (Khavari, P. A., et al, J Intern Med, 252(1):1-10 (2002)). Wepresent evidence here that the exogenously expressed cell cycleregulator, p21^(WAF-1/Cip-1), delivered via a recombinant adenovirusattenuates proliferative responses associated with excessive scarring.With appropriate design and application schedule, p21^(WAF-1/Cip-1) hastherapeutic application in disorders of the skin such as keloids andhypertrophic scars where the pathophysiology stems from dysregulation ofproliferative response.

Example 2

This example illustrates rAd-p21 delivery and p21^(WAF-1/Cip-1)expression in wounds.

rAd-p21 gene delivery and expression over time was characterized in therabbit ear excessive scar model after a single intradermal injection asfollows: Treatment Endpoint Time Points for Group (Intradermal Dose)Analysis Analysis 1 vPBS PCR, RT-PCR, 8 hours; Days 1, 3, Morphology 5,7, 10 and 14 2 rAd-Empty Morphology Days 1, 3 and 10 (2 × 10¹⁰ PN/wound)3 rAd-p21 PCR, RT-PCR 8 hours; Days 1, 3, (2 × 10⁶ PN/wound) Morphology5, 7, 10 and 14 4 rAd-p21 PCR, RT-PCR 8 hours; Days 1, 3, (2 × 10¹⁰PN/wound) Morphology 5, 7, 10 and 14Methods:

Two to four, 6-mm diameter wounds were induced per rabbit ear. Samplesize for analysis consisted of three to eight wounds per treatmentgroup. Two wounds from identical wounding positions on both ears werepooled and placed in a single tube to meet the tissue requirements forRT-PCR and PCR assays. This tube represented the average of two woundsand one assay sample. For PCR and RT-PCR assays at harvest time points 8hours, days 1, 3, 5, 7 and 10, N=1 or 4 representing 2 or 8 woundstotal. For day 14, one wound was placed in a single tube as one sample,therefore N=1 or 4 representing 1 or 4 wounds total per group. Formorphological evaluation, one wound represented one sample and two tofour samples were evaluated per group.

A vPBS treatment group served as vehicle control for both PCR /RT-PCRassays and morphological evaluation. rAd-Empty treatment served ascontrol for adenoviral effects in this study.

Test Reagent Preparation: rAd-p21 and rAd-Empty: Stock virus was dilutedon day 0 of the study in vPBS diluent and maintained on ice. The dilutedviruses were brought to room temperature a half hour prior tointradermal injection into animals.

Rabbit Preparation: Female, New Zealand White rabbits were anesthetizedwith an intramuscular injection of 70 mg/kg of Ketamine, 5 mg/kg ofXylazine, and 0.1 mg/kg of Butorphanol. A hair depilator (Nair™) wasapplied to ears to remove hair and ears were rinsed with warm tap waterand the surgical area was scrubbed with Betadine and isopropanol.Animals were transferred from the pre-operating room to the operatingroom.

Surgical Procedures and Treatments: Under sterile conditions, two tofour, 6mm wounds were made with a Trephine on the ventral side of eachear to the depth of the cartilage. The cartilage and overlying skin wereremoved with a hemostat from each wound. Wound marginal areas wereprepared with Mastisol® and the wounds were covered with OpSite®dressing to prevent drying. Dressings remained on the wounds for theduration of the study. 2×10⁶ or 2×10¹⁰ PN per wound of rAd-p21 wereinjected intradermally around wound marginal areas in a total volume of100 μL per wound using a 28 G ½ insulin injection syringe. Injectionsites on wounds were in positions 3, 6, 9 and 12 o'clock and within 2-3mm of the wound margin areas. Procedures were repeated on both ears forall rabbits in groups 1, 3 and 4. One ear was wounded per rabbit in therAd-Empty treatment group at days 1, 3 and 10 time points. Animals werereturned to cages and allowed food and water ad libitum.

Endpoint Analysis: At specified sacrifice time points, animals wereeuthanized with an overdose of Euthasol CIII 200 mg/kg, iv and therabbit ears were amputated at the base. Full-thickness wounds wereexcised with a 10 mm tissue biopsy punch and placed in a microcentrifugetube containing 250 μL of QIAGEN RNAlater™ (RNA stabilization reagent).All tissues were immersed in the RNA stabilization reagent and stored at4° C. for PCR and RT-PCR analysis. On the day 14 time point, allprocedures were the same as above except one wound from an identical earwound position was placed in a microcentrifuge tube, representing onesample. For morphological evaluation, wounds were bisected, and half ofthe wound was frozen in Optimum Cryosection Temperature Compound (OCT)and the remaining half was fixed in 4% paraformaldehyde at 4° C. for 4hours, transferred to 70% EtOH, and processed for Trichromrie staining.Morphological examination was performed on wound tissue sections forinflammation and wound healing responses.

Quantitative PCR/Quantitative RT-PCR and Preparation of Standard Curve:Quantitative PCR and RT-PCR (QPCR and QRT-PCR) procedures were used toquantify rAd-p21 DNA and transgene expression as previously described byWen et al., Exp Eye Res. 77(3):355-65 (2003). DNA and RNA wereco-extracted from approximately 50-100 mg of tissue using Tri-Reagent®.

Results:

rAd-p21 delivery and human p21^(WAF-1/Cip-1) gene expression wascharacterized over time in the rabbit ear excessive scar model after asingle intradermal dose was delivered immediately after wounding. QPCRand QRT-PCR techniques were implemented to quantify rAd-p21 DNA andhuman p21^(WAF-1/Cip-1) gene expression in the wound area.

QPCR and QRT-PCR Evaluation: 2×10⁶ or 2×10¹⁰ PN per wound of rAd-p21 wasdelivered by a single intradermal injection to rabbit ear wounds. EachrAd-p21 analyzed sample was 2×10⁶ or 2×10¹⁰ PN per each of two woundscombined thus totaling 4×10⁶ or 4×10¹⁰ total PN, respectively. On day14, there was only one wound per sample.

The highest rAd-p21 DNA levels were observed at 8 hours and one day fromboth the low and the high rAd-p21 dose groups. DNA levels decreased overa 14 day period by over 1.0 and 3.0 logs in the low and the high doserAd-p21 groups, respectively. The highest rAd-p21 RNA level was observedat day 3 post intradermal injection in the high dose group. RNA levelsdropped approximately 0.5-1.0 log by days 7, 10 and 14 as compared topeak levels at day 3. The RNA levels in the low dose rAd-p21 group wereundetectable (Below Quantifiable levels;BQL) 8 hours and one day aftertreatment. The onset of detectable RNA expression in the low doserAd-p21 group was observed on day 3. Peak RNA levels were observed ondays 3 and 14 with no significant difference between day 3 and day 14 inthe low dose group (p<0.5; Fisher's Post Hoc ANOVA). There wereapproximately 0.5-1.0 log lower RNA levels at days 5, 7 and 10 whencompared to the RNA levels on days 3 and 14 in the low dose rAd-p21group. As expected, all vPBS samples were negative confirming no crosscontamination of samples or cross reactivity of human p21 primersequence with endogenous rabbit sequence. This RNA expression profile atthe low dose over time demonstrates a similar expression trend as thehigh dose rAd-p21 group.

Morphological Evaluation: The morphological changes in rabbit woundsfollowed typical phases of acute wound healing processes. Briefly, at 8hours post-wounding, inflammatory cell infiltration in wounds wasobserved. Inflammatory cell influx increased in all wounds on days 1 and3. By day 5, granulation tissue started to fill the wound beds andepithelial migrating tongues were observed around the wound edges. Atdays 7, 10 and 14, wound beds were filled with granulation tissue andcovered by epithelium. Thinner granulation tissue and epithelial layerswere noticed in the low rAd-p21 dose treatment group when compared tothe remaining groups at days 10 and 14. However, the low dose rAd-p21group showed denser cellularity than the high dose group at day 14. Thisdata suggests that rAd-p21 can attenuate the volume of granulationtissue and the thickness of epithelium in the wound scar.

Discussion:

Human rAd-p21 DNA delivery and p21^(WAF-1/Cip-1) RNA expression weredetected in both high and low doses of rAd-p21 treatment groups in therabbit ear wounds. p21^(WAF-1/Cip-1) RNA levels were maintained over a14 day period in both the high and low dose groups.

Example 3

This example illustrates that rAd-p21 treatment inhibits scar thickness.

An excessive scar rabbit model was used to determine the effect ofrAd-p21 treatment on scar thickness. Enhanced scarring was induced byinjections of PDGF-BB (2 μg) protein into a rabbit ear wound asdescribed previously. A second injection of 2×10¹⁰ PN of either rAd-p21or rAd-Empty followed seven days later. Scar height was measured as aresponse to treatment and effects were maximally observed atapproximately 11 days post rAd-p21 treatment. Scar tissue was measuredbetween day 18 and 35 (post initial wounding) and tissue was harvestedon day 35 after the initial PDGF-BB injection.

FIG. 5 demonstrates that rAd-p21 treatment attenuates scar thicknessafter intradermal delivery in the rabbit ear excessive scar model. Thisdata supports previous observations that in the normal scar environmentlow doses of rAd-p21, for example, 2×10⁶ PN per wound (7×10⁶ PN/cm²) areefficacious in reducing scar height in this model. When PDGF-BB is usedto induce enhanced scar formation, more rAd-p21 is required to overcomethese scar effects.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method for reducing scarring, the method comprising administering apolynucleotide comprising an expression cassette to skin of a subjecthaving a wound, wherein the expression cassette comprises a promoteroperably linked to a polynucleotide encoding p21^(WAF1/Cip1).
 2. Themethod of claim 1, wherein the DNA is administered as part of a vector.3. The method of claim 2, wherein the vector is a viral vector.
 4. Themethod of claim 3, wherein the viral vector is an adenoviral vector. 5.The method of claim 4, wherein the adenoviral vector is a replicationdeficient adenoviral vector.
 6. The method of claim 1, wherein theadministrating step results in decreased keloids or hypertophic scarringat the wound compared to scarring on an untreated wound.
 7. The methodof claim 4, wherein the adenoviral vector is administered at a dose ofbetween 10⁵ and 10⁹ particle number (PN) per cm² of the wound.
 8. Themethod of claim 1, wherein the vector is administered in a biocompatiblematrix.
 9. The method of claim 8, wherein the matrix comprisescollagenous, metal, hydroxyapatite, bioglass, aluminate, bioceramicmaterials, purified proteins or extracellular matrix compositions. 10.The method of claim 8, wherein the matrix is a collagen matrix.
 11. Themethod of claim 1, wherein the skin is burned.
 12. A pharmaceuticalcomposition comprising an expression cassette and a pharmaceuticallyacceptable excipient, wherein the composition is suitable for topicaladministration and the expression cassette comprises a promoter operablylinked to a polynucleotide encoding p21^(WAF1/Cip1).
 13. Thepharmaceutical composition of claim 12, wherein the expression cassetteis within a biocompatible matrix.
 14. The pharmaceutical composition ofclaim 12, wherein the matrix comprises a viral vector comprising theexpression cassette.
 15. The pharmaceutical composition of claim 14,wherein the viral vector is an adenoviral vector.
 16. The pharmaceuticalcomposition of claim 15, wherein the adenoviral vector is a replicationdeficient adenoviral vector.
 17. The pharmaceutical composition of claim12, wherein the matrix comprises collagenous, metal, hydroxyapatite,bioglass, aluminate, bioceramic materials, purified proteins orextracellular matrix compositions.
 18. The pharmaceutical composition ofclaim 12, wherein the matrix is a collagen matrix.